Chromosome segregation at mitosis and meiosis is essential for all cellular reproduction. Disruptions in this process have profound consequences for human health. Centromeres specify the assembly of a unique set of functional elements on the chromatin fiber that provide motility, sister chromatid cohesion and mechanosensory mechanisms that link these functions to cell cycle checkpoints. CENP-A is a centromere specific homologue of the core nucleosomal protein histone H3. The specific assembly of CENP-A at human centromeres requires a combination of protein-protein interactions within the nucleosome, differentiated DNA contact sites at the surface of the nucleosome and a unique regulatory pathway, leading to a hypothesis for selective recognition of centromeric DNA by CENP-A histone complexes. CENP-A nucleosomes will reconstituted with centromeric DNA sequences isolated by affinity for CENP-A. Analysis of DNA-protein interactions will reveal whether CENP-A exhibits specific recognition of centromeric DNA. The assembly of CENP-A into chromatin will be examined to test the hypothesis that CENP-A chromatin assembly occurs through a novel, non-S phase pathway. CENP-A associated proteins will be isolated and characterized to explore its functional interactions. Mutational approaches will combine long term transfection methods with novel assay methods for centromere motility, mechanical properties and spatial organization in living cells.